Dayu Tian,Lan Huang,Zhao Zhang,Zhenfeng Tian,Shaolin Ge,Chenghui Wang,Yonghua Hu,Ying Wang,Jun Yang
Cellulose is an important component of tobacco ( Nicotiana tabacum L.) cell walls, which can be precursors for many harmful compounds in smoke. Traditional cellulose content analysis methods involve sequential extraction and separation steps, which are time-consuming and environmentally unfriendly. In this study, a novel method was first introduced to analyze cellulose content in tobacco via two-dimensional heteronuclear single quantum coherence (2D HSQC) NMR spectroscopy . The method was based on derivatization approach to allow the dissolution of insoluble polysaccharide fractions of tobacco cell walls in DMSO‑ d 6 /pyridine- d 5 (4:1 v/v) for NMR analysis. The NMR results suggested that besides the main NMR signals of cellulose, partial signals of hemicellulose including mannopyranose , arabinofuranose, and galactopyranose units could also be identified. In addition, the utilization of relaxation reagents has proved to be an effective way to improve the sensitivity of 2D NMR spectroscopy, which was beneficial for quantification of biological samples with limited quantities. To overcome the limitations of quantification using 2D NMR, the calibration curve of cellulose with 1,3,5-trimethoxybenzene as internal reference was constructed and thus the accurate measurement of cellulose in tobacco was achieved. Compared with the chemical method, the interesting method was simple, reliable, and environmentally friendly, which provided a new insight for quantitative determination and structure analysis of plant macromolecules in complex samples.
Wanwan Wang,Jibao Cai,Zhenyu Xu,Yi Zhang,Fanchao Niu,Miao Gao,Jun Yang
A method was developed for rapid qualitative determination of lignocellulose in the tobacco cell wall by utilizing 2D heteronuclear single quantum coherence NMR spectra (2D HSQC NMR). Traditional methods for analyzing the structure of lignocellulose involve many steps of separation and extraction, which is labor-intensive. In this work, the whole cell wall was milled and dissolved in deuterium solvent. The solvent dimethylsulfoxide (DMSO-d 6 ) containing hexamethylphosphoramide (HMPA-d 18 ) enhanced swelling of the sample and gave high-resolution spectra. The tobacco samples are ball milled at different ball milling times, and the state of the particles is observed through an electron microscope, and then the probability of the particles being less than 5 µm is counted. Through the comparison of the abundance and integration of the peak signals in the spectra under different transmittances, it was determined that when the milling time was 6 h, the quality of the NMR spectra was the best. The optimum conditions of characterizing tobacco structure were DMSO-d 6 /HMPA-d 18 solution and 6 h milling time. Under these conditions, complete representation of the structure of lignocellulose and simplified process could be achieved. Graphical abstract
Weijie Lin,Dengwen Ning,Dezhong Xu,Shuai Wu,Qiuxia Zou,Ajoy Kanti Mondal,Fang Huang
This study compares the behavior of combined and free lignin on the cellulose enzymatic hydrolysis (EH). The combined lignin refers to the lignin naturally existed in the lignocellulose and the free lignin refers to the cellulolytic enzyme lignin. Three kinds of lignocelluloses (i.e., Moso bamboo, horsetail pine and aspen) are chosen for the research. Lignin is mainly biosynthesized of guaiacyl (G), syringyl (S), and p -hydroxyphenyl (H) structural units. The HSQC NMR analysis shows that the free lignin of Moso bamboo, horsetail pine and aspen are mainly composed of G-S-H, G-H and G-S type lignin units, respectively. Furthermore, a blood glucose meter (BGM), UV and fluorescence spectrophotometer are used to determine the cellulose conversion, enzyme adsorption and fluorescence intensities in the EH. It shows that the combined lignin has a weaker inhibitory effect on the EH breakdown than the free lignin. The influence of EH follows the order of Moso bamboo > aspen > horsetail pine. This study can be applied to the development of theoretical guidelines for mitigating the residual prohibition of lignin in the lignocellulose EH.
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